DAP-kinase related protein

ABSTRACT

A new protein, which is a novel homologue of DAP-kinase, has been isolated. This novel calmodulin-dependent kinase is a cell death-promoting protein functioning in the biochemical pathway which involves DAP (death-associated protein)—kinase (e.g., forming a cascade of sequential kinases, one directly activating the other). Alternatively, the two kinases may operate to promote cell death in parallel pathways.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the national stage entry of internationalApplication No. PCT/US99/13411, filed Jun. 15, 1999, which claims thebenefit of Provisional Application No. 60/089,294, filed Jun. 15, 1998,the entire contents of which are hereby incorporated by reference. Thepresent Application is also related to and hereby incorporates byreference the entire contents of application Ser. No. 08/810,712 filedMar. 3, 1997 now U.S. Pat. No. 6,160,106 issued Dec. 12, 2000 andapplication Ser. No. 08/631,097, filed Apr. 12, 1996 now U.S. Pat. No.5,968,816 issued Oct. 19, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a DAP-kinase related protein.

2. Description of the Related Art

One of the factors which determines the proliferation state of cells isthe balance between the growth-promoting effects of proto-oncogenes andthe growth-constraining effects of tumor-suppressor genes. One mechanismby which tumor-suppressor genes exert their growth-constraining effectis by inducing a cell to undergo a physiological type of death. Such acontrolled cell death is evident in a multitude of physiologicalconditions including metamorphosis, synaptogenesis of neurons, death oflymphocytes during receptor repertoire selection, and controlledhomeostasis in the bone marrow and other proliferative tissues, etc.This cell death is regulated by the interaction of the cell with othercells or with cell products, for example through the activity ofsuitable cytokines.

Growth-inhibiting cytokines have a double effect on the target cell.They can either inhibit the proliferation of the cell and/or give riseto cell death. To date, blockage or activation of expression of knowntumor-suppressor genes was shown to counteract or enhance, respectively,cytokines inhibition of cells growth (Kimchi, 1992) but did not have anyeffect on the death-promoting action of cytokines. For example, thegrowth inhibitory response to cytokines, such as TGF-β, was markedlyreduced by the inactivation of the Rb gene, or the response to IL-6 wasenhanced by introducing activated p53 genes (Pietenpol et al, 1990; Levyet al, 1993).

Apoptosis is a genetically controlled cell death process which isimportant in various developmental stages, as well as for cellmaintenance and tissue homeostasis (Jacobson et al., 1997). During thelast few years, many of the key players in this process have beenidentified, including receptors, adaptor proteins, proteases, and otherpositive and negative regulators (Green et al., 1998; White, 1996). Oneof the positive mediators of apoptosis, which has been cloned by thepresent inventors, is DAP-kinase (Deiss et al., 1995). This protein wasdiscovered by a functional approach to gene cloning, based ontransfections of mammalian cells with anti-sense cDNA libraries andsubsequent isolation of death-protective cDNA fragments (Deiss et al.,1995; Deiss et al., 1991; Kimchi, 1998; Kissil et al., 1998;Levy-Strumpf et al., 1998). The anti-sense cDNA of DAP-kinase protectedHeLa cells from interferon-gamma-induced cell death, and this propertyserved as the basis for its selection.

DAP-kinase is a calcium/calmodulin-regulated 160 kDa serine/threonineprotein kinase associated with actin microfilaments (Deiss et al., 1995;Cohen et al., 1997). Its structure contains at least two additionaldomains that might mediate interactions with other proteins: ankyrinrepeats, and a typical death domain located at the C-terminal part ofthe protein (Deiss et al., 1995; Cohen et al., 1997). Overexpression ofDAP-kinase in various cell lines results in cell death, and thisdeath-promoting effect of DAP-kinase depends on at least three features:the catalytic activity, presence of the death domain, and the correctintracellular localization (Cohen et al., 1997; Cohen et al., 1999).Several independent lines of evidence proved that DAP-kinase is involvedin apoptosis triggered by different external signals, includinginterferon-γ, TNF-α, activated Fas receptors, and detachment of cellsfrom the extracellular matrix (Deiss et al., 1995; Cohen et al., 1997;Cohen et al., 1999; Inbal et al., 1997). A tumor suppressive functionwas recently attributed to the DAP-Kinase, coupling the control ofapoptosis to metastasis (Inbal et al., 1997).

So far, only a few serine/threonine kinases were implicated in theregulation of programmed cell death, either as death-promoting anddeath-protecting proteins (Anderson, 1997; Bokoch, 1998). One suchcandidate is the JNK/SAPK (Basu et al., 1998). In one example, it wasshown to mediate apoptosis induced by detachment from extracellularmatrix (named anoikis) (Cardone et al, 1997). In this system, the JNKpathway is activated by MEKK-1, whose kinase activity is stimulated bycaspase cleavage (Cardone et al., 1997). JNK may antagonize BCL-2anti-apoptotic effects by phosphorylation (Park et al., 1997; Maundrellet al., 1997).

Another serine/threonine kinase is RIP, which like DAP-Kinase alsopossesses the death domain. RIP was shown to positively mediateapoptosis in cell cultures (Stanger et al., 1995). However, in vivostudies in RIP-deficient mice demonstrated its ability to exertanti-apoptotic effects by mediating the TNF-α-induced TNF-β activation(Kelliher et al., 1998). Other RIP members, RIP2 and RIP 3 were alsorecently identified and shown to possess pro-apoptotic effects (McCarthyet al., 1998; Sun et al., 1998; Yu et al., 1999).

Among the negative regulators of apoptosis is the protein kinase AKT.This protein was shown to phosphorylate BAD and thereby to prevent itfrom complexing and blocking the anti-apoptotic activity of BCL-X_(L)(Datta et al, 1997; del Peso et al., 1997). AKT was also recently shownto phosphorylate pro-caspase-9, thus blocking its normal processing(Cardone et al., 1998).

Recently, the isolation and characterization of novel kinase members,homologous in their catalytic domains to DAP-kinase, was reported (Kawaiet al., 1998; Kogel et al., 1998; Sanjo et al., 1998). One protein,named ZIP-kinase, was found to be 80% identical to DAP-kinase within thekinase domain, yet it lacks the CaM-regulatory domain and the otherdomains and motifs characteristic of DAP-kinase. Zip-kinase contains aleucine zipper domain at the C-terminus and is localized to the nucleus(Kawai et al., 1998; Kogel et al. 1998). The activation of ZIP kinaseoccurs by a different mechanism involving homo-dimerization, mediated byits leucine zipper domain. However, unlike DAP-kinase, ZIP-kinase is anuclear protein, which instead of being regulated by acalmodulin-binding domain, is activated by homo-dimerization of itsleucine-zipper motifs (Kogel et al., 1998). Another two less conservednuclear proteins, DRAK1 and DRAK2, which are closely related to eachother, and which share 50% identity with the kinase domain ofDAP-kinase, were also recently characterized. Like ZIP-kinase, the DRAK1and DRAK2 proteins also lack the CaM-regulatory domain. Theoverexpression of these two proteins in NIH3T3 cells induces somemorphological changes associated with apoptosis, dependent on thefunctionality of their kinase domain (Sanjo et al., 1998). Togetherthese kinases form a novel subfamily of serine/threonine kinases, as isevident from multiple sequence and phylogenetic analysis (Inbal et al.,1999).

Ectopic expression of the three wild type kinases, but not theircatalytically inactive mutants, induced morphological changescharacteristic of apoptosis (Kawai et al., 1998; Sanjo et al., 1998).Yet, in the case of ZIP-Kinase, these results are still controversial(Kogel et al., 1998).

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto the content or a date of any document is based on informationavailable to the applicant at the time of filing and does not constitutean admission as to the correctness of such a statement

SUMMARY OF THE INVENTION

A new protein, DAP-Kinase-related 1 protein (DRP-1), which is a novelhomologue of DAP-kinase, has been isolated. This novelcalmodulin-dependent kinase is a 42 kDa serine/threonine kinase whichshows a high degree of homology to DAP-kinase both in its catalyticdomain and its calmodulin-regulatory region. The catalytic domain ofDRP-1 is also homologous to recently identified ZIP-kinase and, to alesser extent, to the catalytic domains of DRAK1/2.

DRP-1 is localized to the cytoplasm as shown by immunostaining andcellular fractionation assays. In vitro kinase assays indicate that wildtype DRP-1, but not a kinase inactive mutant, undergoesautophosphorylation and phosphorylates an external substrate in aCa2+/CaM-dependent manner. Ectopically expressed DRP-1 is able to induceapoptosis in various types of cells; with this killing being dependenton its kinase activity. The dominant negative form of DAP-Kinase (DAPkDD) is a potent blocker of apoptosis induced DRP-1. Thus, DRP-1 may be adeath-promoting protein functioning in the biochemical pathway whichinvolves DAP (death-associated protein)-kinase (e.g., forming a cascadeof sequential kinases, one directly activating the other).Alternatively, the two kinases may operate to promote cell death inparallel pathways.

The present invention provides for a DRP-1 protein and functionalhomologues thereof having at least 85% sequence identity to the DRP-1sequence of SEQ ID NO:2. Also provided is a fragment of DRP-1, whicheither is capable of inducing cell death or lacks such capability butinstead is capable of inhibiting the activity of DRP-1 or a functionalhomologue thereof to induce cell death, and a homologous fragment whichhas at least 85% sequence identity thereto and which has the sameproperties.

The present invention further provides an isolated DNA molecule encodingfor such DRP-1 protein, functional homologues thereof, or fragmentsthereof. Also included within the scope of the present invention areisolated DNA molecules which hybridize to the nucleotide sequenceencoding DRP-1 protein under moderately or highly stringent conditionsand encode a calmodulin-dependent serine/threonine kinase having theproperty of being capable of inducing cell death.

Other further aspects of the present invention include a compositioncomprising the DRP-1 protein, functional homologues and fragmentsthereof, and an antibody which specifically recognizes DRP-1 but doesnot cross-react with DAP kinase or ZIP kinase.

Yet another aspect of the present invention is directed to a singlestranded RNA molecule complementary to at least a portion of the mRNAencoding the DRP-1 protein of SEQ ID NO:2. This single strandedantisense RNA molecule can be used in a method of neutralizing DRP-1mRNA by hybridizing to the DRP-1 mRNA to prevent its translation intoDRP-1 protein.

The present invention also provides a method for screening individualsfor predisposition to cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:2)sequence of the DAP-kinase homologue, DRP-1. The initiation (ATG) andstop (TAA) codons are boxed. The polyadenylation signal (ATTAAA) isunderlined. The kinase domain and the calmodulin regulatory regions arein bold or underlined by a dash, respectively.

FIGS. 2A–2B show the multiple sequence alignment of the serine/threoninekinase domains (FIG. 2A) of the DAP-kinase-related proteins, DAP-kinase(SEQ ID NO:3), ZIP-kinase (SEQ ID NO:4), DRP-1 (corresponding toresidues 13–275 of SEQ ID NO:2), DRAK1 (SEQ ID NO:5) and DRAK 2 (SEQ IDNO:6), conducted according to Hanks and Quinn (1991) with identicalamino acids boxed and homologous amino acids shown with gray shading,and the multiple sequence alignment of the calmodulin regulatory regions(FIG. 2B) of DAP-kinase (SEQ ID NO:7), DRP-1 (corresponding to residues292 to 320 of SEQ ID NO:2), smMLCK (SEQ ID NO:8), CaMKIIa (SEQ ID NO:9),CaMKI (SEQ ID NO:10), CaMKIV (SEQ ID NO: 11), and ZIP-Kinase (SEQ ID NO:12) conducted manually, keeping the conserved (boxed) regions aligned toeach other. The corresponding region of ZIP-Kinase which does notcontain homology to DAP-Kinase and ZIP-Kinase CAM-regulatory regions isgiven at the bottom of FIG. 2B.

FIG. 3A shows Northern blot analysis of polyA+RNA extracted from variouscell lines for mRNA expression of DRP-1, FIG. 3B show Western blotanalysis of in vitro transcription and translation of DRP-1, and FIG. 3Cshows protein expression of DRP-1 in HeLA cells on an immunoblot.

FIGS. 4A and 4B show control COS-7 cells and cellular localization ofDRP-1 in COS-7 cells, respectively, and FIG. 4C shows a Western blot offractions from a detergent extraction of COS-7 cells transfected with apcDNA3 vector expressing either FLAG-tagged DRP-1 or DAP-Kinase.

FIG. 5A shows in vitro kinase activity of DRP-1 and FIG. 5B shows aWestern blot of DRP-1 proteins.

FIGS. 6A–6B show fluorescent microscope images of 293 cells transfectedby pcDNA3-luciferase as a negative control (FIG. 6A), by pcDNA3-ΔCaMDAP-Kinase as positive control (FIG. 6B), by pCDNA-DRP-1 (FIG. 6C), andby pCDNA3-K42A DRP-1 (FIG. 6D). Apoptotic cells are indicated by arrows.

FIG. 7 shows the scores of apoptotic cells in a graph of the percentageof apoptotic cells resulting from the transfections of FIGS. 6A–6D.

FIGS. 8A and 8B show DRP-1 protein expression in 293 transfected cellsin immunoblots to anti-FLAG antibodies (FIG. 8A) and anti-vinculinantibodies (FIG. 8B).

FIG. 9A shows that DAP kinase death domain protects from DRP-1 inducedapoptosis, and FIG. 9B shows an immunoblot of DRP-1 protein expressionin 293 transfected cells.

FIG. 10A shows a schematic representation of a series of generateddeletion mutant, and FIG. 10B shows an immunoblot containing extracts of293 cells transiently transfected with GFP and the series of deletionmutants, (DRP-1 fragments, cloned in pCDNA3, and tagged with HA epitopeat the C-terminus), as in FIGS. 8A and 8B are probed with anti-HAantibodies for DRP-1 detection and anti-vinculin antibodies toquantitate the loaded protein amounts. In FIG. 10B, pCDNA3²-luciferaseis the negative control.

FIG. 11A shows fluorescent microscope images of the transientlytransfected cells of FIG. 10B, and FIG. 11B shows a graph of the scorein percent apoptotic cells in FIG. 11A resulting from co-transfectionsof 293 cells with 1–2 μg HA-tagged wild type DRP-1 or various deletionmutants of DRP-1 after 24 hours (average S.D. calculated fromtriplicates of 100 cells each).

FIGS. 12A and 12B show by Western analysis that the C-terminal part ofDRP-1 is required for its homo-dimerization. In FIG. 12A, wild typeDRP-1 is shown to undergo specific homo-dimerization. The lanescorrespond to the following co-transfections (5 μg of DRP-1 constructsand 20 μg of RFX1-ΔSmaI constructs/9 mm plate): (1)DRP-1-FLAG+RFX1-ΔSmaI-HA (control to rule-out nonspecific attachment ofDRP-1 to HA beads or to an irrelevant gene). (2)RFX-ΔSmaI-FLAG+DRP-1-HA(control to rule out nonspecific attachment ofDRP-1 to FLAG beads or to an irrelevant gene). (3) DRP-1−FLAG+DRP-1-HA.Both IP directions and their Western blottings are shown. In FIG. 12B,truncation of C-terminal 40 amino acids of DRP-1 is shown to abolish itshomo-dimerization. The lanes correspond to the followingco-transfections (5 μg of each construct/90 mm plate): (1)DRP-1-FLAG+DRP-1-HA (2) DRP-1-FLAG+DRP-1-Δ40-HA (3)DRP-1-FLAG+DRP-1-Δ73-HA (4) DRP-1-FLAG+DRP-1-Δ85-HA. The lower panelquantitate the immunoprecipitation efficiency of DRP-1-FLAG by theanti-FLAG antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery by the present inventorof a novel serine/threonine kinase with remarkable homology to thecatalytic and CaM-regulatory domains of DAP-kinase. This kinase, namedDAP-kinase-related protein 1 (DRP-1), is a 42 kDa cytoplasmic proteinwhich exhibits minor associations with insoluble matrix elements. Thenucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequences of thisDRP-1 protein are shown in FIG. 1. It is composed of 1742 nucleotides.The predicted initiation and stop codons are boxed, and thepolyadenylation signal is underlined. The protein kinase domain is shownin bold letters and corresponds to amino acid residues 13 to 275 of SEQID NO:2. This protein displays 80% identity with the catalytic domain ofDAP-kinase. The calmodulin-regulatory region is underlined with a dashedline; this region displays high homology to the corresponding region inDAP-kinase. The remainder of the C-terminal short part of the protein(the last 40 amino acid residues corresponding to residues 321 to 360 ofSEQ ID NO:2)differs completely from DAP-kinase. Thus, DAP-kinase-relatedI protein does not carry all of the other motifs and protein modulescharacteristic of DAP-kinase. The mRNA expression levels transcribedfrom this gene are low.

Another protein, ZIP-kinase, which by virtue of its sequence homology tothe kinase domain of DAP-Kinase, is also a member of theDAP-Kinase-related proteins subfamily, was recently identified (Kawai etal., 1998; Kogel et al., 1998). Unlike DAP-Kinase and DRP-1, ZIP-kinaseis a nuclear protein, which instead of being regulated by acalmodulin-binding domain, is activated only by homo-dimerization viaits leucine-zipper motifs (Kawai et al., 1998). To this group ofkinases, another two less homologous nuclear proteins, DRAK1 and DRAK2,were recently added (Sanjo et al., 1998). Together they form a novelsubfamily of serine/threonine kinases, as is evident from multiplesequence and phylogenetic analyses (Inbal et al., 1999). A multiplesequence alignment of the kinase domain of these serine/threoninekinases is shown in FIG. 2A.

To check the cellular functions of DRP-1, the laboratory of the presentinventor overexpressed wild type DRP-1 in various cell lines and foundthat it induced apoptosis as measured by a few parameters. Unlike thewild type DRP-1, a kinase inactive mutant of DRP-1 (DRP-1 K42A), did notinduce apoptosis, although it was expressed at a similar level in thetransfected cells. In vitro kinase assays confirmed that DRP-1 K42A isindeed unable to phosphorylate MLC or under autophosphorylation. Also, atruncated form of DRP-1 which lacks the CaM-regulatory region, could beshown to induce very high levels of apoptosis, in a similar way to theanalogous truncation of the CaM-regulatory region of DAP-Kinase (ΔCaM;Cohen et al., 1997). Such dependence on the catalytic activity for theapoptotic function is apparent also in the other members ofDAP-kinase-related proteins (Kawai et al., 1998; Sanjo et al., 1998).

In a deletion mutant study, which is also presented in the Exampleherein, the existence of a positive element responsible for apoptoticinduction which is located at the C-terminal part of DRP-1 is confirmed.This C-terminal tail of DRP-1 is also essential for its dimerization.Thus, self-dimerization is a requirement for the functionality of thiskinase in apoptotic assays, although this property can be overrided by afurther deletion of the CaM-regulatory region. Like DRP-1,ZIP-kinase-induced cell death is also controlled by its ability toundergo homo-dimerization via the C-terminal leucine zipper domain(Kawai et al., 1998). Three point mutations in the leucine zipper domainabolished the homo-dimerization as well as the ability of ZIP-kinase toundergo autophosphorylation in vitro and significantly reduced itsability to induce cell death of NIH 3T3 cells. It seems reasonable toassume that activation of these kinases is achieved by homo-dimerizationfollowed by trans-phosphorylation events.

The high homology in the kinase domains of DAP-kinase and DRP-1, and thefinding that they are both localized to the cytoplasm (either in solubleor insoluble forms), imply that they may share the same or closelyrelated substrates. The phosphorylation sites for these kinases on thesubstrate may be either different or identical. Thus, these kinases maycooperate to induce apoptosis in the same cell type or, alternatively,function independently in different cell types, tissues or organs, or inresponse to different stimuli or time windows. Another possibility isthat these kinases act sequentially along the same signaling pathway toinduce apoptosis.

The present invention thus provides for the polypeptide of DRP-1 and fora calmodulin-dependent serine/threonine kinase homologue having theproperties of DRP-1, such as the ability to phosphorylate protein in acalcium/calmodulin dependent manner and the ability to induce programmedcell death or apoptosis, and having at least 85% sequence identity tothe amino acid sequence SEQ ID NO:2 of DRP-1. Preferably, thecalmodulin-dependent serine/threonine kinase homologue has at least 90%sequence identity, and more preferably, at least 95% sequence identityto SEQ ID NO:2.

The term “sequence identity” as used herein means that the amino acidsequences are compared by alignment according to Hanks and Quinn (1991)with a refinement of low homology regions using the Clustal-X program.Such an amino acid alignment is shown in FIGS. 2A and 2B where theidentical amino acid residues are presented in boxes (cutoff=50%) andhomologous amino acid residues, determined according to the PAM 250matrix, are presented by gray shading (cutoff=65%).

The Clustal-X program referred to in the previous paragraph is theWindows interface for the ClustalW multiple sequence alignment program(Thompson et al., 1994). The Clustal-X program is available over theinternet at ftp://ftp-igbmc.u-strasbg.fr/pub/clustalx/. Of course, itshould be understood that if this link becomes inactive, those ofordinary skill in the art can find versions of this program at otherlinks using standard internet search techniques without undueexperimentation. Unless otherwise specified, the most recent version ofany program referred herein, as of the effective filing date of thepresent application, is the one which is used in order to practice thepresent invention.

If the above method for determining “sequence identity” is considered tobe nonenabled for any reason, then one may determine sequence identityby the following technique. The sequences are aligned using Version 9 ofthe Genetic Computing Group's GDAP (global alignment program), using thedefault (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of−12 (for the first null of a gap) and a gap extension penalty of −4 (pereach additional consecutive null in the gap). After alignment,percentage identity is calculated by expressing the number of matches asa percentage of the number of amino acids in the claimed sequence.

In addition to the full length polypeptide of DRP-1 or a functionalhomologue thereto with at least 85% sequence identity, the presentinvention also provides for a fragment of the DRP-1 protein of SEQ IDNO:2 which either maintains the ability to induce cell death or lacksthis ability but instead is capable of inhibiting the cell killingability of DRP-1 protein or its functional homologue described above. Itwas unexpectedly discovered by the present inventor that the 40 aminoacid C-terminal tail (residues 321 to 360 of SEQ ID NO:2) is critical toinduction of cell death. As the action of DRP-1 is dependent ondimerization, the 40 amino acid tail, by itself, can inhibit the abilityof DRP-1 to induce cell death by interfering with and preventing DRP-1from dimerizing. Furthermore, it was also unexpectedly discovered thatthe catalytic domain, by itself (without the calmodulin regulatorydomain and the 40 amino acid C-terminal tail, e.g., amino acid residues13 to 275 of SEQ ID NO:2), is super-killing. One of ordinary skill inthe art can readily obtain fragments of the full length sequence of thepresent invention using N-terminal amino peptidases or C-terminalcarboxypeptidases. Each fragment can then be readily tested to see if itpossesses one of the two functions described herein for such fragments,without undue experimentation.

Besides fragments of DRP-1 having the above-mentioned properties,fragments having an amino acid sequence with at least 85% sequenceidentity to the above fragments of DRP-1, preferably with at least 90%sequence identity, and more preferably with at least 95% sequenceidentity, and maintaining the cell death induction or inhibitionproperties of the original fragment, are also comprehended by thepresent invention.

Also comphrended by the present invention are chemical derivatives ofthe DRP-1 and functional homologues and fragments thereof, as definedabove, where a “chemical derivative” contains additional chemicalmoieties not normally part of the DRP-1 amino acid sequence. Covalentmodifications of the amino acid sequence are included within the scopeof this invention. Such modifications may be introduced into DRP-1 orfragments thereof by reacting targeted amino acid residues of thepeptide with an organic derivatizing agent that is capable of reactingwith selected side chains or terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxylmethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo-beta-(5-imidazoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl-2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5–7.0 because this agent is relatively specific for the histidylside chain. Parabromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino acid-containing residues includeimidoesters, such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methyliosurea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione, andninhydrin. Derivatization of arginine residues requires that thereaction be performed in alkaline conditions because of the high pKa ofthe guanidine functional group. Furthermore, these reagents may reactwith the groups of lysine, as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidazole andtetranitromethane are used to form O-acetyl tyrosyl species and e-nitroderivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′N—C—N—R′) such as1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)] carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

The present invention also comprehends an isolated DNA molecule whichincludes a nucleotide sequence encoding the DRP-1 protein of SEQ IDNO:2, a functional homologue thereof as described above, or a fragmentof DRP-1 which either maintains the ability of DRP-1 to induce celldeath or lacks this ability but is instead capable of inhibiting thecell killing ability of DRP-1 protein, as defined above. The isolatedDNA molecule according to the present invention is also intended tocomprehend a DNA molecule which hybridizes under moderately stringent,preferably highly stringent, conditions to the nucleotide sequenceencoding DRP-1 (corresponding to nucleotides 62 to 1141 of SEQ ID NO:1)and which encodes a polypeptide which maintains the cell death inductionproperties of DRP-1. The present invention further comprehends isolatedDNA molecules which hybridize under moderately stringent, preferablyhighly stringent, conditions to a nucleotide sequence which encodes fora fragment of DRP-1 which either maintains the ability of DRP-1 toinduce cell death (i.e., nucleotides 98 to 886 of SEQ ID NO:1 encodingthe catalytic kinase domain of DRP-1) or lacks the ability but isinstead capable of inhibiting the cell killing ability of DRP-1 protein(i.e., nucleotides 1022 to 1141 of SEQ ID NO:1 encoding the 40 aminoacid C-terminal tail of DRP-1). Furthermore, polypeptides encoded by anynucleic acid, such as DNA or RNA, which hybridizes to the nucleotidesequence of nucleotides 62 to 141 of SEQ ID NO:1 under moderatelystringent or highly stringent conditions are considered to be within thescope of the present invention as long as the encoded polypeptidemaintains the ability of DRP-1 to induce cell death.

As used herein, stringency conditions are a function of the temperatureused in the hybridization experiment, the molarity of the monovalentcations and the percentage of formamide in the hybridization solution.To determine the degree of stringency involved with any given set ofconditions, one first uses the equation of Meinkoth et al. (1984) fordetermining the stability of hybrids of 100% identity expressed asmelting temperature Tm of the DNA—DNA hybrid:Tm=81.5° C.+16.6(LogM)+0.41(% GC)−0.61(% form)−500/Lwhere M is the molarity of monovalent cations, % GC is the percentage ofG and C nucleotides in the DNA, % form is the percentage of formamide inthe hybridization solution, and L is the length of the hybrid in basepairs. For each 1° C. that the Tm is reduced from that calculated for a100% identity hybrid, the amount of mismatch permitted is increased byabout 1%. Thus, if the Tm used for any given hybridization experiment atthe specified salt and formamide concentrations is 10° C. below the Tmcalculated for a 100% hybrid according to the equation of Meinkoth,hybridization will occur even if there is up to about 10% mismatch.

As used herein, “highly stringent conditions” are those which provide aTm which is not more than 10° C. below the Tm that would exist for aperfect duplex with the target sequence, either as calculated by theabove formula or as actually measured. “Moderately stringent conditions”are those which provide a Tm which is not more than 20° C. below the Tmthat would exist for a perfect duplex with the target sequence, eitheras calculated by the above formula or as actually measured. Withoutlimitation, examples of highly stringent (5–10° C. below the calculatedor measured Tm of the hybrid) and moderately stringent (15–20° C. belowthe calculated or measured Tm of the hybrid) conditions use a washsolution of 2×SSC (standard saline citrate) and 0.5% SDS (sodium dodecylsulfate) at the appropriate temperature below the calculated Tm of thehybrid. The ultimate stringency of the conditions is primarily due tothe washing conditions, particularly if the hybridization conditionsused are those which allow less stable hybrids to form along with stablehybrids. The wash conditions at higher stringency then remove the lessstable hybrids. A common hybridization condition that can be used withthe highly stringent to moderately stringent wash conditions describedabove is hybridization in a solution of 6×SSC (or 6×SSPE (standardseline-phosphate-EDTA)), 5× Denhardt's reagent, 0.5% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA at a temperature approximately20° to 25° C. below the Tm. If mixed probes are used, it is preferableto use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel,1987, 19989.

Additional aspects of the present invention are vectors which carry theisolated DNA molecule according to the present invention and a host cellwhich is transformed with the isolated DNA according to the presentinvention.

The present invention further provides for antisense RNA complementaryto at least a portion of a messenger RNA (mRNA or “sense” RNA) moleculewhich is the transcription product of the DNA sequence encoding theDRP-1 protein of SEQ ID NO:2. The antisense DRP-1 sequence can bechemically synthesized or it can be expressed in host cells. However,when expressed in host cells, the expressed antisense RNA must be stable(i.e., does not undergo rapid degradation). Moreover, the antisenseDRP-1 RNA, will essentially specifically only hybridize to the senseDRP-1 mRNA and form a stable double-stranded RNA molecule that isessentially non-translatable. In other words, the antisense DRP-1 RNAprevents the expressed sense DRP-1 mRNA from being translated intoactive DRP-1 protein. When expressed in host cells, a vector-borneantisense DRP-1 sequence may carry either the entire DRP-1 gene sequenceor merely a portion thereof as long as the antisense DRP-1 sequence iscapable of hybridizing to “sense” DRP-1 mRNA to prevent its translationinto DRP-1 protein. Accordingly, an “antisense” sequence of the presentinvention can be defined as a sequence which is capable of specificallyhybridizing to “sense” DRP-1 mRNA to form a non-translatabledouble-stranded RNA molecule.

The antisense DRP-1 sequence need not hybridize to the entire length ofthe DRP-1 mRNA. Instead, it may hybridize to selected regions, such asthe 5′-untranslated sequence, the coding sequence, or the3′-untranslated sequence of the “sense” mRNA. In view of the size of themammalian genome, the antisense DRP-1 sequence is preferably at least17, more preferably at least 30, base pairs in length. However, shortersequences may still be useful, i.e., they either fortuitously do nothybridize to other mammalian sequences, or such “cross-hybridization”does not interfere with the metabolism of the cell in a manner and to adegree which prevents the accomplishment of an object of this invention.The greater the length of the antisense sequence and the greater thenumber of complementary base pairs, the greater the number ofnon-complementary bases that can be tolerated, especially if thenon-complementary bases are scattered. Both the preferred hybridizationtarget on DRP-1 and the preferred antisense sequence length are readilydetermined by systematic experiment.

Standard methods such as described in Sambrooke et al., (1989) can beused to systematically remove an increasingly larger portion of theantisense DRP-1 sequence from a plasmid vector. Besides the full lengthantisense DRP-1 sequence, a series of staggered deletions may begenerated, preferably at the 5′-end of the antisense DRP-1 sequence.This creates a set of truncated antisense DRP-1 sequences that stillremain complementary to preferably the 5′-end of the sense DRP-1 mRNAand as a result, still forms a RNA molecule that is double-stranded atthe 5′-end of the sense DRP-1 mRNA (complements the 3′-end of anantisense DRP-1 RNA) and remains non-translatable.

The antisense RNA according to the present invention can be used in amethod to neutralize a mRNA molecule, which is the transcription productof the DNA sequence encoding the DRP-1 protein of SEQ ID NO:2, byallowing the antisense RNA to hybridize to the DRP-1 mRNA to prevent itstranslation into DRP-1 protein.

A further aspect of the present invention is directed to a composition,such as a pharmaceutical composition, which contains DRP-1, functionalhomologues or fragments thereof and a pharmaceutically-acceptableexcipient, carrier, diluent, or auxiliary agent.

An antibody, which specifically recognizes DRP-1 or functionalhomologues thereof is part of the present invention as long as theantibody does not cross-react with DAP-Kinase or ZIP-kinase. Forinstance, an antibody that specifically recognizes the unique 40 aminoacid C-terminal tail of DRP-1, which is not present in DAP-Kinase orZIP-kinase, is a preferred embodiment of the antibody according to thepresent invention. Such an antibody can be used for diagnostic imaging,purification of DRP-1 etc.

The terms “antibody” or “antibodies” as used herein are intended toinclude intact antibodies, such as polyclonal antibodies or monoclonalantibodies (mAbs), as well as proteolytic fragments thereof such as theFab or F(ab′)₂ fragments. Furthermore, the DNA encoding the variableregion of the antibody can be inserted into other antibodies to producechimeric antibodies (see, for example, U.S. Pat. No. 4,816,567) or intoT-cell receptors to produce T-cells with the same broad specificity(Eshhar et al., 1990; Gross, et al., 1989). Single chain antibodies canalso be produced and used. Single chain antibodies can be single chaincomposite polypeptides having antigen binding capabilities andcomprising a pair of amino acid sequences homologous or analogous to thevariable regions of an immunoglobulin light and heavy chain (linkedV_(H)–V_(L) or single chain F_(V)). Both V_(H) and V_(L) may copynatural monoclonal antibody sequences or one or both of the chains maycomprise a CDR-FR construct of the type described in U.S. Pat. No.5,091,513 (the entire contents of which are hereby incorporated hereinby reference). The separate polypeptides analogous to the variableregions of the light and heavy chains are held together by a polypeptidelinker. Methods of production of such single chain antibodies,particularly where the DNA encoding the polypeptide structures of theV_(H) and V_(L) chains are known, may be accomplished in accordance withthe methods described, for example, in U.S. Pat. Nos. 4,946,778,5,091,513 and 5,096,815, the entire contents of each of which are herebyincorporated herein by reference.

As mentioned above, the terms “antibody” or “antibodies” are also meantto include both intact molecules as well as fragments thereof, such as,for example, Fab and F(ab′)₂, which are capable of binding antigen. Faband F(ab′)₂ fragments lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody (Wahl et al., 1983). It will beappreciated that Fab and F(ab′)₂ and other fragments of the antibodiesuseful in the present invention may be used for the detection andquantitation of DRP-1 or functional homologues thereof according to themethods used for intact antibody molecules. Such fragments are typicallyproduced by proteolytic cleavage, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab)₂ fragments).

The present invention comprehends not only the intact antibodies orfragments, but also any molecule which includes an antigen bindingportion of an antibody such that the molecule is capable of binding tothe antigen. It is well within the skill of the art for the artisan tomake e.g., fusion proteins which include antigen binding portions of anantibody fused to any other material which is desired to be carried tothe antigen binding site, such as marker molecules, toxins, etc.

The antibodies, or fragments of antibodies, of the present invention maybe used to quantitatively or qualitatively detect the presence of DRP-1or functional homologues according to the present invention in a sample.The antibody according to the present invention may also be used for theisolation and purification of DRP-1 or homologues and fragments thereof,such as in an affinity column where the antibodies are immobilized on asolid phase support or carrier.

By “solid phase support or carrier” is intended any support capable ofbinding antigen or antibodies. Well-known supports, or carriers, includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Those skilled in the artwill know many other suitable carriers for binding antibody or antigen,or will be able to ascertain the same by use of routine experimentation.

One of the ways in which the DRP-1-specific antibody can be detectablylabeled is by linking the same to an enzyme and used in an enzymeimmunoassay (EIA). This enzyme, in turn, when later exposed to anappropriate substrate, will react with the substrate in such a manner asto produce a chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. The detection canbe accomplished by to calorimetric methods which employ a chromogenicsubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect DRP-1 protein through theuse of a radioimmunoassay (RIA) (Chard, T., “An Introduction toRadioimmune Assay and Related Techniques” (In: Work, T. S., et al.,Laboratory Techniques in Biochemistry in Molecular Biology, NorthHolland Publishing Company, New York (1978), incorporated by referenceherein). The radioactive isotope can be detected by such means as theuse of a gamma counter or a liquid scintillation counter or byautoradiography. Radioactively labeled antibodies or antibody fragmentscan also be used for their capacity to kill cells bound by suchantibodies, or cells in the immediate vicinity which are exposed to theradiation from such antibodies. It is also possible to label theantibody with a fluorescent compound, a chemiluminescent orbioluminescent compound.

The antibody molecules of the present invention may also be adapted forutilization in an immunometric assay (also known as a “two-site” or“sandwich” assay) which is well know in the art.

In the present specification, the term “programmed cell death” is usedto denote a physiological type of cell death which results fromactivation of some cellular mechanisms, i.e., death which is controlledby the cell's machinery. Programmed cell death may, for example, be theresult of activation of the cell machinery by an external trigger, e.g.,a cytokine, which leads to cell death. The term “apoptosis” is also usedinterchangeably with programmed cell death.

The term “tumor” in the present specification denotes an uncontrolledgrowing mass of abnormal cells. This term includes both primary tumors,which may be benign or malignant, as well as secondary tumors, ormetastases, which have spread to other sites in the body.

DRP-1 can be used to inhibit growth and metastasis of tumors. Tumorcells are exposed to a variety of death-inducing signals which, incombination with DAP-kinase-related I, can lead to death of the tumorcells. For example, in the blood stream, invading tumor cells mustresist programmed cell death that is induced by interactions withcytotoxic T lymphocytes, natural killer cells, and macrophages, and withthe cytokines which these hematopoietic cells secrete (e.g., IFNs, TNF,IL-1β). Tumor cells must also resist the apoptotic cell death induced bynitric oxide anions produced by the endothelial cells, and withstandmechanical shearing forces caused by hemodynamic turbulence. Moreover,during the intravasation or extravasation processes, and during growthin a foreign hostile microenvironment, locally produced inhibitorycytokines (e.g., TGF-β or loss of cell-matrix interactions (e.g.,detachment from the basement membranes) also trigger apoptotic celldeath.

DRP-1 is useful in promoting death of tumor cells. The protein may beadministered to patients, in particular, to cancer patients, whichadministration may cause death of the tumor cells. The protein may beadministered per se, or may be administered by an expression vectorcomprising a DNA molecule of the present invention.

Because DRP-1 displays 80% identity with the catalytic domain ofDAP-kinase and has a region which displays a high homology to thecalmodulin-regulatory region of DAP-kinase, it is expected that DRP-1has enzymatic kinase activity, which is calmodulin-dependent. Thus,DRP-1 has use as an enzyme and may be used, for example, as the enzymein any in vitro enzymatic reaction which requires the presence of akinase enzyme. Accordingly, DRP-1 can be used in vitro to catalyzephosphorylation reactions as a kinase.

DRP-1 is capable of inducing apoptotic cell death when overexpressed invarious cell lines. This ectopic cell death is blocked specifically bythe death domain of DAP-kinase, suggesting possible crosstalk betweenthese two kinases. Thus, DRP-1 may also be used for promoting the deathof normal or tumor cells and for suppressing the metastatic activity oftumor cells. A particular application of the death-promoting aspect isin therapy of diseases or disorders associated with uncontrolled,pathological cell growth, e.g., cancer (primary tumors and metastasis),psoriasis, autoimmune disease and others. Indeed, it is expected thatthe DAP-kinase-related protein I of the present invention and DNAencoding it, may be used in the same manner as disclosed in detail inU.S. application Ser. Nos. 08/810,712 and 08/631,097, as well as WO95/10630.

According to a further aspect of the present invention, referred toherein at times as “the screening aspect”, DRP-1 DNA molecules are usedin order to screen individuals for predisposition to cancer. Inaccordance with this aspect the screening is carried out by comparingthe sequence of each of the DAP-kinase-related I DNA molecules to eachof the respective DAP genes in the individual, or by following RNAand/or protein expression. The absence of a DAP-kinase-related I gene, apartial deletion or any other difference in the sequence that indicatesa mutation in an essential region, or the lack of a DRP-1 RNA and/orprotein which may result in a loss of function may lead to apredisposition for cancer. For screening, preferably a battery ofrelated DAP and DRP-1 genes maybe used, as well as different antibodies.

In the screening aspect, DAP-kinase related product I molecules may alsobe used for prognostic purposes. For example, if a tumor cell lacksDRP-1 activity, this may reflect high chances of developing metastasis.In addition, DRP-1 positive cells may be more susceptible to control bychemotherapeutic drugs that work by inducing apoptosis, so that thechoice of treatment modalities may be made based upon the DRP-1 state ofthe cells.

The DAP-kinase-related product can be used to screen individuals forpredisposition to cancer. There is provided a method for detecting theabsence of a DRP-1 gene, a partial deletion or a mutation (i.e., pointmutation, deletion or any other mutation) in the DRP-1 genes of anindividual, or the absence of a DRP-1 RNA or protein, comprising probinggenomic DNA, cDNA, or RNA from the individual with a DNA probe or amultitude of DNA probes having a complete or partial sequence of theDRP-1 genes, or probing protein extracts with specific antibodies.

A particular application of the screening aspect of this invention is inthe screening for individuals having a predisposition to cancer, anabsence of the gene, or a detected mutation or deletion indicating thatthe individual has such a predisposition.

One example of a method in accordance with the screening aspecttypically comprises the following steps:

-   -   (a) obtaining a sample of either genomic DNA from cells of the        individual or cDNA produced from mRNA of said cells;    -   (b) adding one or more DNA probes, each of said probes        comprising a complete or partial sequence of a DRP-1 gene;    -   (c) providing conditions for hybridization to determine whether        the DRP-1 gene is present or absent, i.e., whether there is a        match between the sequence of the DNA probe or probes and a        sequence in the DNA of said sample or a mismatch, a mismatch        indicating a deletion or a mutation in the endogenous DNA and a        predisposition to cancer in the tested individual.

Other examples of the screening aspect of the invention are well knownto the skilled artisan and include, but are not limited to, Northernblots, RNase protection assays, and various PCR procedures.

The mutation in the DRP-1 gene, indicating a possible predisposition tocancer, can also be detected by the aid of appropriate antibodies whichare able to distinguish between a mutated, a non-functional and a normalfunctional DRP-1 gene product. In addition, mutations that abolishprotein translation or transcription due to promoter inactivation can bedetected with the aid of antibodies that are used to react with proteincell extracts. Screening is also possible with respect to metastases.

Having now generally described the invention, the same will be morereadily understood through reference to the following example which isprovided by way of illustration and is not intended to be limiting ofthe present invention.

EXAMPLE

In this study, the identification and the structure/function analysis ofa novel DAP-kinase-related protein, DRP-1, is described, DRP-1 is a 42kDa Ca²⁺/CaM-regulated serine/threonine kinase which shows high degreeof homology to DAP (Death Associated Protein)-kinase. The homology spansover the catalytic domain and the calmodulin-regulatory region, whereasthe rest C-terminal part of the protein differs completely fromDAP-kinase and displays no homology to any known protein. The catalyticdomain is also homologous to the recently identified ZIP-kinase and to alesser extent to the catalytic domains of DRAK1/2, thus forming togethera novel subfamily of serine/threonine kinases. DRP-1 is localized to thecytoplasm as shown by immunostaining and cellular fractionation assays.In vitro kinase assays indicate that wild type DRP-1, but not a kinaseinactive mutant, undergoes autophosphorylation and phosphorylates anexternal substrate in a Ca²⁺/CaM-dependent manner. Ectopically expressedDRP-1 is able to induce apoptosis in various types of cells. Cellkilling by DRP-1 is dependent on two features: the intact kinaseactivity and the presence of C-terminal 40 amino acids shown to beinvolved in self-dimerization of the kinase. Interestingly, furtherdeletion of the CaM-regulatory region overrided the indispensable roleof the C-terminal tail and generated a “super-killer” mutant. Finally, adominant negative fragment of DAP-kinase encompassing the death domainis a potent blocker of apoptosis induced by DRP-1. This implies apossible functional connection between DAP-kinase and DRP-1. Theexperiments conducted in this study and the results obtained arepresented below.

Materials and Methods

cDNA Cloning and Northern Blot Analysis

A PCR fragment of 364 bp was obtained from a λgtll human spleen cDNAlibrary (Clontech) using primers from the deduced DRP-1 sequence,1047-GGCCGGATGAGGACCTGAGG-1066 (SEQ ID NO:13) and1411-TCCACACTCCCACCCCAGACTC-1390 (SEQ ID NO:14). To obtain the fulllength cDNA of DRP-1, the same cDNA library was screened with theradiolabeled PCR product. Positive phage clones were isolated, cDNA wassubcloned into a BlueScript vector and analyzed by restriction enzymemapping and DNA sequencing. A 270 bp 3′-fragment from the full lengthcDNA of DRP-1 was generated by EcoRI digestion, and used to probe polyA+RNA prepared by a standard procedure from various cell lines.

In Vitro Transcription and Translation Assay

The full length cDNA was used as a template for in vitro transcriptionfrom the T7 promoter. This RNA was translated in reticulocyte lysate(TNT® T7 Quick Coupled Transcription/Translation System; Promega) byconventional procedures, with [³⁵S] methionine (Amersham) as a labeledprecursor. The reaction product was then run on 12% SDS-PAGE gel,followed by sodium salicylate incubation for signal amplification. Thegel was dried and exposed to X-ray film.

In vitro Kinase Assay

293 cells were transfected by a FLAG-tagged wild type DRP-1, DRP-1 K42Amutant, or mock-transfected. Cell lysates of 293 transfected cells wereprepared as previously described (Deiss et al., 1995).Immunoprecipitation of DRP-1 or DRP-1 K42A mutant from 150 μg totalextract was done with 20 μl anti-FLAG M2 gel (IBI, Kodak) in 500 μl ofPLB supplemented with protease and phosphatase inhibitors for 2 h at 4°C. Following three washes with PLB, the immunoprecipitates were washedonce with reaction buffer (50 mM HEPES pH 7.5, 20 mM MgCl₂, and 0.1mg/ml BSA). The proteins bound to the beads were incubated for 15 min at30° C. in 50 μl of reaction buffer containing 15 μCi [γ32p] ATP (3pmole), 50 μM ATP, 5 μg MLC (Sigma), and where indicated, also 1 μMbovine calmodulin (Sigma), 0.5 mM CaCl₂, or 3 mM EGTA in the absence ofcalmodulin/CaCl₂. Protein sample buffer was added to terminate thereaction, and after boiling, the proteins were analyzed on 11% SDS-PAGE.The gel was blotted onto a nitrocellulose membrane and ³²P_labeledproteins were visualized by autoradiography.

Immunostaining of Cells

DRP-1 transfected or mock-transfected COS-7 cells were plated on glasscover-slips (13 mm diam.). After 48 hours, the cells werefixed/permeabilized in 3% formaldehyde for 5 min, methanol 5 min,acetone 2 min. The cells were blocked in 10% NGS for 30 min andincubated with anti-FLAG antibodies (dilution 1:100; IBI, Kodak) in 10%NGS for 60 min. Rhodamine-conjugated goat anti-mouse secondaryantibodies (dilution 1:200; Jackson Immuno Research Lab.) and thenucleic acid dye, Oligreen (dilution 1:5000; Molecular Probes), fornuclear staining were then applied. The coverslips were mounted inMowiol and observed under fluorescence microscope.

Detergent Extraction Assay

Sub-confluent cultures of COS-7 transfected cells, grown on 9 cm plate,were washed once with PBS and then with MES buffer (50 mM MES pH 6.8,2.5 mM EGTA, 2.5 mM MgCl₂). The cells were extracted for 3 min with 0.5ml of 0.5% Triton X-100 in MES buffer supplemented with proteaseinhibitors. The supernatant (the soluble fraction-Sol) was collected,centrifuged for 2 min. at 16,000x g at 4° C., and the clear supernatantwas then transferred to new tubes. Two volumes of cold ethanol wereadded and the tubes were incubated at −20° C. for overnight, centrifuged10 min. at 16,000x g at 4° C. and resuspended in 200 μl of 2× proteinsample buffer without dye. The detergent insoluble matrix (InSol)remaining on the plate was extracted in 200 μl of 2× protein samplebuffer, scraped from the plate with rubber policeman and collected intotube. The samples were loaded on 10% SDS-PAGE, 100 μg protein extractswere loaded on each lane from the Sol fraction, equivalent volumes ofInSol were loaded. Analysis of the proteins was done using monoclonalanti-FLAG antibodies (dilution 1:200; IBI, Kodak).

Cell Lines, Transfections and Apoptotic Assays

All cell lines were grown in DMEM (Biological Industries) supplementedwith 10% fetal calf serum (Bio-Lab). For transient transfection, 1×10⁵cells per well, were seeded in a 6 well plate a day before transfection.Transfections were done by calcium-phosphate method. For cell deathassays by inducing overexpression, a mixture containing 1.5 μg of celldeath plasmid (expressing either DRP-1 or ΔCaM DAPk mutant) and 0.5 μgof pEGFP-NI plasmid (Clontech) was used. For cell death protectionassays we used a mixture containing 1.2 μg of cell death inducingplasmid (either DRP-1 or ΔCaM DAPk mutant), 0.5 μg of a plasmid to betested for cell death protection (expressing DAPk-DD, DN FADD orluciferase as negative control), and 0.5 μg of pEGFP-NI plasmid. Cellswere counted and photographed 24 hours after transfection. In eachtransfection, three fields, each consisting of at least 100 GFP-positivecells, were scored for apoptotic cells according to their morphology.When indicated, cell lysates were prepared from the transienttransfection at 24 hours, for protein analysis. The transfections of Ratembryo fibroblasts (REF) and FACS analysis of transfected fibroblastsfor DNA content distribution were done as previously described (Kissilet al., 1998).

Co-Immunoprecipitation Assays

293 cells grown in 90 mm plates (1×10⁶ cells/plate) were co-transfectedwith 5 μg FLAG-tagged or HA-tagged DRP-1 and 20 μg of HA-tagged orFLAG-tagged RFX1ΔSmaI, respectively, or with DRP-1-HA and DRP-1-FLAG, 5μg each. Immunoprecipitation of DRP-1 or RFX1-ΔSmaI from 1 mg totalextract was done using anti-FLAG M2 gel or anti-HA as described above.Detection of bound proteins was done using anti-HA antibodies (dilution1:1000, Babco) or anti-FLAG antibodies. For the deletion mutant study, 5μg of FLAG-tagged fully length DRP-1 were co-transfected with 5 μg ofHA-tagged DRP-1 deletion mutants. Immunoprecipitation of DRP-1 from 1 mgtotal extract was done using anti-FLAG M2 gel as described above.Detection of co-immunoprecipitated proteins (the mutants of DRP-1 orfull length DRP-1) was done using anti-HA antibodies.

Nucleotide Sequence Accession Number

The nucleotide sequence of human DRP-1 has been submitted to theGenBank™/EBI Data Bank (accession no. AF052941). The murine DRP-1 isalso deposited at the GenBank™/EBI Data Bank (accession no. AF052942).

Results

Cloning of DRP-1 To identify proteins that share homologous sequenceswith DAP-kinase, EST databases were searched using the BLAST™ program.Two ESTs of human and murine origin showed remarkable amino acidhomology to the catalytic domains of DAP-kinase and the recentlyidentified protein ZIP-kinase (79.5% and 80.2% identity, respectively).A second EST search was performed using the 5′ and the 3′ ends of thehuman EST, which identified a few more overlapping ESTs. A putativenovel cDNA sequence was generated and used to design primers for cloningthe full length cDNA. PCR performed on human spleen cDNA libraryamplified a 364 bp fragment that was further used to screen the samelibrary. The full length cDNA was then isolated, subcloned intoBlueScript vector, and sequenced.

The isolated cDNA was found to be 1742 bp long and to contain aserine/threonine kinase domain with all of the 12 characterizedsubdomains present (Park et al., 1997, FIG. 1A). Sequence alignmentindicated that the catalytic domain of DRP-1 has 80% sequence identityto that of DAP-kinase and ZIP-kinase, yet less 50% sequence identity tothe newly identified DRAK proteins (FIG. 2A). Like DAP-kinase but unlikeZIP-kinase, DRP-1 carries a typical CaM-regulatory region adjacent toits catalytic domain, as shown in FIGS. 1 and 2B. As compared with otherkinases such as CaKIIa and MLCK, DRP-1 has the highest homology toDAP-kinase in this region, as shown in FIG. 2B. The remaining shortstretch of amino acids at the C-terminal part of DRP-1 (40 amino acidtail) displays no homology to any known protein.

Expression of DRP-1 and Tissue Distribution

To check the RNA expression of DRP-1, polyA+RNA was prepared fromvarious cell lines and hybridized to a probe designed from the lessconserved region of DRP-1. A single weak band of 1.9 kb appeared in somecell lines, in a Northern blot analysis of poly A+RNA (3 micrograms)extracted from various cell lines (FIG. 3A), suggesting that the mRNA isexpressed at low amounts in HeLa, 293 and MCF-7 cells. The mRNA washybridized with a radiolabeled human DRP-1 probe. The position of thetranscript is indicated by an arrow. From PCR analysis of various cDNAlibraries and the data gathered from EST searches, it was concluded thathuman DRP-1 is expressed, at least, in spleen, colon, breast, andleukocyte tissue.

In vitro transcription and translation assays conducted in reticulocytelysates using the cloned DRP-1 cDNA as a template generated a singleprotein band of about 42 kDa in size, as predicted by its sequence. Thisprotein band, obtained by Western blot analysis of in vitro transcribedand then translated DRP-1, is shown in FIG. 3B. A FLAG-tagged DRP-1 wasthen cloned into pCDNA3 vector and expressed in HeLa cells. A protein of42 kDa was evident upon immunoblot analysis of the cell lysates withanti-FLAG antibodies, shown in FIG. 3C. In this case, 24 hours followingtransfection, the cells were harvested and lysed. The extracted proteinswere separated by SDS-PAGE and then immunoblotted with anti-FLAGantibodies.

Cellular Localization of Ectopically Expressed DRP-1

In order to follow the cellular localization of the exogenous DRP-1,FLAG-tagged DRP-1 was expressed in COS-7 cells. COS-7 cells weretransfected by a FLAG-tagged DRP-1 cloned in pCDNA3 vector, fixed andpermeabilized in 1% formaldehyde followed by methanol/acetone treatment.Cells were visualized under fluorescence microscope. Immunoblot analysisproved that DRP-1 was expressed in these cells. For the immunostainingprocedure, the non-transfected (FIG. 4A) and DRP-1 transfected (FIG. 4B)COS-7 cells were then fixed and reacted both with Oligreen for nuclearstaining and anti-FLAG antibodies for DRP-1 staining. Specific DRP-1staining was detected in the cytoplasm of these cells, as shown in FIG.4B.

A gentle cell extraction was performed with nonionic detergent, 0.5%TRITON X-100, that removes lipids and soluble proteins, leaving intactthe detergent insoluble matrix composed of the nucleus, the cytoskeletonframework, and cytoskeleton-associated proteins. In contrast toDAP-kinase, which is exclusively localized to the cytoskeleton, andhence found only in detergent insoluble fractions (Cohen et al., 1997,FIG. 4C), DRP-1 was preferentially eluted from the detergent solublefraction, while a small amount was eluted from the insoluble fraction,as shown in FIG. 4C. Thus, it was concluded that DRP-1 is a cytoplasmicprotein with minor association with insoluble matrix components.

Intrinsic Kinase Activity of DRP-1

To test whether DRP-1 functions as a kinase as predicted from the aminoacid sequence, an in vitro kinase assay was performed using myosin lightchain (MLC) as an exogenous substrate. This substrate was chosen becauseit is phosphorylated by DAP-kinase (Cohen et al., 1997). DRP-1 wastransfected into human kidney 293 cells, immunoprecipitated, andincubated with MLC in the presence and absence of Ca2+ and calmodulin.Both MLC phosphorylation and DRP-1 autophosphorylation were evident, ascan be seen from FIG. 5A.

In assaying the in vitro kinase activity of DRP-1, the proteins wereassayed in the presence or absence of CA2+/CaM and MLC. The proteinswere run on 11% SDS-PAGE and blotted to nitrocellulose membrane. FIG. 5Ashows the autophosphorylation of DRP-1 and MLC phosphorylation,respectively, as seen after exposure of X-ray film. FIG. 5B shows theDRP-1 proteins by incubation of the same blot with anti-FLAG antibodiesand ECL detection.

The addition of Ca2+/calmodulin to the reaction mixture increased theamount of phosphorylated MLC, in accordance with the assumption that,like DAP-kinase, DRP-1 is negatively regulated by the autoinhibitorycalmodulin binding domain, and that this inhibition is removed by thebinding of Ca2⁺ calmodulin. A catalytically inactive mutant of DRP-1,DRP-1 K42A, did not phosphorylate MLC and failed to undergoautophosphorylation even though higher amounts of DRP-1 protein werepresent, as can be seen from FIG. 5A. Thus, DRP-1 was found to functionin vitro as a kinase that is capable of phosphorylating itself and anexternal substrate. This latter property is stimulated by the additionof Ca2+ and calmodulin.

DRP-1 Induces Apoptosis in a Variety of Cell Lines

The high homology to DAP-kinase in the kinase and calmodulin-bindingregions suggested the value of checking whether DRP-1 is involved inapoptosis. The wild type DRP-1 and the catalytically inactive mutant ofDRP-1, DRP-1 K42A, which are cloned in pCDNA3 vector, were transfectedinto 293 cells. To quantitate the number of apoptotic cells, theseconstructs were transfected with a vector expressing the GFP protein.The GFP protein was used as a marker to visualize the transfected cellsand to assess the apoptotic frequency among the transfectants accordingto morphological alterations. Apoptotic cells were scored after 24hours. Overexpression of the DRP-1 resulted in massive apoptotic celldeath (50–60%), as compared to the basal level of apoptotic cells causedby transfection of the non-relevant gene luciferase, shown in FIGS.6A–6B and 7.

Most of the GFP positive green cells rounded up and shrunk; some of themshowed cytoplasmic blebs, and some were further fragmented into“apoptotic bodies.” In addition, some of the transfected cells detachedfrom the plate. This apoptotic cell death was only slightly lower thanthat of an activated DAP-kinase mutant lacking the autoinhibitorycalmodulin regulatory region (ΔCaM; apoptotic values of 70–80%). Incontrast, when the cells were transfected with the kinase inactivemutant of DRP-1, DRP-1 K42A, as shown in FIGS. 6A–6D and 7, no apoptosiswas observed. This experiment was repeated six times with reproducibleresults.

Western blot analysis of transfected cells, using anti-FLAG antibodies,confirmed the expression of both the exogenous wild type and K42A mutantof DRP-1 (FIGS. 8A and 8B). Similar results were also observed in humanSV-80 fibroblasts. In another type of assay, the effect of ectopicallyexpressed DRP-1 on the DNA content of rat embryo primary fibroblasts(REF cells) was assessed, as previously described (Kissil et al., 1999).The REFS were co-transfected with DRP-1 and a membrane-bound form of GFPand then after 48 hours subjected to FACS analysis of their DNA content.A fraction of cells displaying a sub-G1 population, indicative of cellscontaining fragmented DNA, appeared exclusively in the DRP-1 transfectedcells but not in cells transfected with a control vector or with theDRP-1 K42A mutant form. No effect was found on cell cycle distributionof the viable cells.

To obtain the results shown in FIGS. 6A–6D, 1×10⁵ 293 cells/well wereco-transfected with FLAG-tagged wild type DRP-1 or K42A mutant of DRP-1,1.5 microgram/well and GFP, 0.5 microgram/well. GFP positive cells werevisualized under fluorescent microscope and scored for the appearance ofapoptotic morphology 24 hours after transfection. Apoptotic cells areindicated by arrows. The fluorescent microscopic images correspond to293 cells transfected by pCDNA3-luciferase as negative control (FIG.6A), pCDNA#-deltaCaM DAP-kinase as positive control (FIG. 6B),pCDNA3-DRP-1 (FIG. 6C), pCDNA3-K42A DRP-1 (FIG. 6D).

In FIG. 7, graphs show the percentage of apoptotic cells resulting fromthe above-mentioned transfections (average±S.D. calculated fromtriplicates of 100 cells each). The scores were taken from the sameexperiment shown in FIGS. 6A–6D.

In FIGS. 8A and 8B, proteins extracted from the transfected cells wereseparated on 10% SDS-PAGE and blotted to nitrocellulose membrane. Theblot was hybridized with anti-FLAG antibodies for DRP-1 detection andanti-vinculin antibodies to quantitate the loaded protein amounts. Theproteins were prepared from the same experiments shown in FIGS. 6A–6D.

DAP Kinase Death Domain Protects From DRP-1 Induced Apoptosis

The structural homology of DRP-1 to DAP-kinase, the common regulation byCa2+/calmodulin, and the finding that both proteins caused apoptosisupon overexpression, suggested that they function along a commonapoptotic pathway. In order to test this possibility, the effect of thedominant-negative DAP-kinase death domain (DAPk DD) on DRP-1-inducedcell death was analyzed. The laboratory of the present inventor showedrecently that overexpression of the fragment encompassing the deathdomain of DAP-kinase acts as a specific dominant-negative mutant,negating the effects of the full length protein (Datta et al., 1997). Asa consequence, it protected cells from TNF-alpha, Fas andFADD/MORTI-induced cell death (Datta et al., 1997).

It has now been discovered that DAPk DD protected cell death induced byDRP-1 in 293 cells. As shown in FIG. 9A, the apoptotic ratio droppedfrom 64.3% to 24.7%. A control transfection including DRP-1 and anon-relevant luciferase DNA excluded the possibility that this blockagewas simply due to larger amount of DNA used in the transfection.Moreover, the effect of DAPk DD was specific, since the death domain ofFADD failed to manifest a similar effect. (FIG. 9A). Western blotanalysis of transfected cells using anti-FLAG antibodies confirmed theexpression of the exogenous DRP-1 in all transfections, as shown in FIG.9B. This experiment was repeated three times with reproducible results.The ability of the death domain of DAP-kinase to block death induced byDRP-1 implies that DAP-kinase and DRP-1 function along a common pathway.

To obtain the results shown in FIG. 9A, 1×10⁵ cells/well of 293 cellswere co-transfected with 1.2 microgram/well of FLAG-tagged wildtypeDRP-1 and 0.5 microgram/well of GFP. The scores are the percentage ofapoptotic cells given as average ±S.D. and calculated from triplicatesof 100 cells each.

To demonstrate the DRP-1 protein expression in 293 transfected cellsshown in FIG. 9B, proteins extracted from the transfected cells wereseparated on 10% SDS-PAGE and blotted to nitrocellulose membrane. Theblot was hybridized with anti-FLAG antibodies for DRP-1 detection andanti-vinculin antibodies to quantitate the loaded protein amounts. Theproteins were prepared from the same experiment shown in FIG. 9A.

Deletion of the C-Terminal Tail of DRP-1 Abolishes its ApoptoticActivity, While Further Truncation of the CaM-Regulatory Region StronglyEnhances the Apoptotic Effect

In order to further understand the mode of DRP-1 action in apoptosis,constructs containing C-terminal truncations of DRP-1 tagged by HA wereconstructed (FIG. 10A). DRP-1 Δ40 lacks the most C-terminal part ofDRP-1 which displays no homology to any known protein. DRP-1 Δ73 lacks,in addition to that, the CaM-regulatory region of DRP-1, and DRP-1 Δ85contains only the catalytic domain. The wild type DRP-1 and the varioustruncation mutants of DRP-1 were transfected into 293 cells. Inductionof apoptotic cell death was assayed as mentioned above in DRP-1 inducedapoptosis. Overexpression of the wild type DRP-1 resulted in apoptosis(25%) while the DRP-1 Δ40 had no effect in these assays. On the otherhand, further truncations of the CaM-regulatory region, yielded mutants(Δ73, Δ85) which acted as “super-killers” (˜90% apoptosis) (FIGS. 11Aand 11B). This experiment was repeated three times with reproducibleresults. Western blot analysis of transfected cells, using anti-HAantibodies confirmed the expression of all DRP-1 forms (FIG. 10B). Thus,the dependence of the apoptotic effect of DRP-1 on its kinase activitywas confirmed again, since removal of the CaM-regulatory region whichacts as an autoinibitory domain generates a constitutively activekinase. In addition, the existence of a positive module in theC-terminal region of DRP-1, which is necessary for its pro-apoptoticeffect, provided that the CaM-regulatory effect is still present, isshown.

The C-Terminal Part of DRP-1 Functions as a Homo-Dimerization Domain

Western analysis performed on proteins extracted from 293 cellstransfected by FLAG-tagged DRP-1 revealed an additional band (notshown). This observation led the present inventor to test whether DRP-1can undergo homo-dimerization. To this aim, two constructs expressingDRP-1 fused to either FLAG or HA tags were co-transfected into 293 cellsand classical pull-down experiments with each one of the two epitopeswere performed. FLAG-tagged DRP-1 could be shown to bind specifically toHA-tagged DRP-1 in both IP directions (FIG. 12A, see lane 3 in both IPPanels). No binding of DRP-1-HA to FLAG beads or to the irrelevantcytoplasmic protein RFX-ΔSmaI could be observed (FIG. 12A, see IPanti-FLAG panel, lanes 2 or 1+2, respectively). Also non-specificbinding of DRP-1-FLAG to HA bead or to RFX-ΔSmaI protein could not bedetected (FIG. 12A, see IP anti-HA panel, lanes 1 or 1+2, respectively).Western analyses confirmed the expression of all proteins in these cellextracts (FIG. 12A, see Western panels).

The observation that a C-terminal truncation of 40 amino acids in DRP-1abolished its apoptotic effect upon ectopic expression in 293 cells,prompted the present inventor to test whether this domain may beinvolved in the homo-dimerization of DRP-1. DRP-1-FLAG was co-expressedin conjugation with the various deletion mutants of DRP-1 tagged by HA.A strong binding of DRP-1-FLAG to the wild type DRP-1-HA was detected,whereas the binding to DRP-1 Δ40 was mostly abolished (FIG. 12B, upperIP panel, compare lane 1 to 2–4). Western analysis confirmed theexpression of wild type DRP-1-HA and all other DRP-1-HA deletion mutantsin these transfections (FIG. 12B, see Western panel). Lower IP panelconfirmed the expression of wild type DRP-1-FLAG in all thesetransfections. Thus, the present inventor concluded that a regionspanning the C-terminal 40 amino acids of DRP-1 is responsible for itshomo-dimerization. This homo-dimerization is probably required for theapoptotic effect of DRP-1, since DRP-1-Δ40 has lost the ability toinduce apoptosis in 293 cells (FIGS. 11A and 11B).

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within know or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent application, issued U.S.or foreign patents, or any other references, are entirely incorporatedby reference herein, including all data, tables, figures, and textpresented in the cited references. Additionally, the entire contents ofthe references cited within the references cited herein are alsoentirely incorporated by reference.

Reference to known method steps, conventional method steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1. An isolated polypeptide, which is a calmodulin-dependentserine/threonine kinase, or a fragment thereof, selected from the groupconsisting of: (A) a polypeptide which is capable of inducing cell death(apoptosis) and comprises the amino acid sequence of SEQ ID NO:2; (B) apolypeptide which has a property of being capable of inducing cell deathand has at least 90% sequence identity to the amino acid sequence of SEQID NO:2; (C) a fragment of a polypeptide of (A) which is capable ofinducing cell death; and (D) a fragment of a polypeptide of (A) whichlacks the property of being capable of inducing cell death and whichinhibits the ability of the polypeptide (A) to induce cell death.
 2. Anisolated DNA molecule comprising a nucleotide sequence encoding thepolypeptide or fragment thereof according to claim
 1. 3. The isolatedDNA molecule according to claim 1, wherein said nucleotide sequenceencodes the amino acid sequence of SEQ ID NO:2.
 4. The isolated DNAmolecule according to claim 3, wherein said nucleotide sequencecorresponds to nucleotides 62 to 1141 of SEQ ID NO:1.
 5. The isolatedDNA molecule according to claim 3, which consists of the nucleotidesequence corresponding to nucleotides 62 to 1141 of SEQ ID NO:1.
 6. Anisolated DNA molecule which hybridizes to the DNA molecule of claim 5under highly stringent conditions and encodes a calmodulin-dependentserine/threonine kinase having the property of being capable of inducingcell death.
 7. A polypeptide capable of inducing cell death, consistingof an amino acid sequence selected from the group consisting of aminoacid residues 13 to 275 of SEQ ID NO:2 and an amino acid sequence havingat least 90% sequence identity to residues 13 to 275 of SEQ ID NO:2. 8.An isolated DNA molecule comprising a nucleotide sequence encoding thepolypeptide of claim
 7. 9. The isolated DNA molecule according to claim8, wherein said nucleotide sequence encodes the amino acid sequencecorresponding to residues 13 to 275 of SEQ ID NO:2.
 10. The isolated DNAmolecule according to claim 9, wherein said nucleotide sequencehybridizes to nucleotides 98 to 886 of SEQ ID NO:1 under highlystringent conditions and encodes a polypeptide capable of inducing celldeath.
 11. A polypeptide capable of inhibiting the ability of thepolypeptide of SEQ ID NO:2 to induce cell death, consisting of an aminoacid sequence selected from the group consisting of amino acid residues321 to 360 of SEQ ID NO:2 and an amino acid sequence having at least 85%sequence identity to residues 321 to 360 of SEQ ID NO:2.
 12. An isolatedDNA molecule consisting of a nucleotide sequence encoding thepolypeptide of claim
 11. 13. The isolated DNA molecule according toclaim 12, wherein said nucleotide sequence encodes the amino acidsequence corresponding to residues 321 to 360 of SEQ ID NO:2.
 14. Theisolated DNA molecule according to claim 13, wherein said nucleotidesequence hybridizes to nucleotides 1022 to 1141 of SEQ ID NO:1 underhighly stringent conditions and encodes a polypeptide capable ofinducing cell death.
 15. A vector comprising the isolated DNA moleculeaccording to claim
 2. 16. A host cell transformed with the isolated DNAmolecule according to claim
 2. 17. A composition comprising apolypeptide according to claim 1 and a pharmaceutically acceptableexcipient, carrier, diluent or auxiliary agent.
 18. A single strandedRNA molecule having 17 to 30 nucleotides in length that is complementaryto at least a portion of the isolated messenger RNA molecule which isthe transcription product of the DNA sequence encoding a polypeptide ofSEQ ID NO:2, wherein said complementary single stranded RNA molecule iscapable of hybridizing to said isolated messenger RNA to prevent itstranslation into said polypeptide of SEQ ID NO:2.
 19. A method ofneutralizing a messenger RNA molecule, which is the transcriptionproduct of the DNA sequence encoding a polypeptide of SEQ ID NO:2,comprising the step of contacting the single stranded RNA molecule ofclaim 18 with the messenger RNA to neutralize the messenger RNA byhybridizing thereto and preventing its translation into the polypeptideof SEQ ID NO:2.
 20. A composition comprising a polypeptide according toclaim 8, and a pharmaceutically acceptable excipient, carrier, diluentor auxiliary agent.
 21. A composition comprising a polypeptide accordingto claim 11, and a pharmaceutically acceptable excipient, carrier,diluent or auxiliary agent.
 22. The polypeptide of claim 1, wherein saidpolypeptide (B) has at least 95% sequence identity to the amino acidsequence of SEQ ID NO:2.
 23. The polypeptide of claim 7 which has atleast 90% sequence identity to residues 13 to 275 of SEQ ID NO:2. 24.The polypeptide of claim 11 which has at least 95% sequence identity toresidues 13 to 275 of SEQ ID NO:2.
 25. The polypeptide of claim 11 whichhas at least 90% sequence identity to residues 321 to 360 of SEQ IDNO:2.
 26. The polypeptide of claim 11 which has at least 95% sequenceidentity to residues 321 to 360 of SEQ ID NO:2.
 27. A vector comprisingthe isolated DNA molecule according to claim
 12. 28. A hostcell-transformed with the isolated DNA molecule according to claim 12.29. A single stranded RNA molecule having 100% complementarity to atleast a portion of the isolated messenger RNA molecule which is thetranscription product of the DNA sequence encoding a polypeptide of SEQID NO:2, wherein the complementary single stranded RNA molecule iscapable of hybridizing to said isolated messenger RNA molecule toprevent its translation into said polypeptide of SEQ ID NO:2.
 30. Amethod of neutralizing a messenger RNA molecule, which is thetranscription product of the DNA sequence encoding a polypeptide of SEQID NO:2, comprising the step of contacting the single stranded RNAmolecule of claim 29 with the messenger RNA to neutralize the messengerRNA by hybridizing thereto and preventing its translation into thepolypeptide of SEQ ID NO:2.